Tooth development is a complex, multistage process that involves reciprocal epithelial-mesenchymal interactions, polarized growth, establishment of bilateral symmetries morphogenetic movements, and cytodifferentiation. These complex developmental events have been described in detail, but the underlying mechanisms of regulation remain largely unclear. In particular, little is known about the nature of the molecules mediating epithelial-mesenchymal interactions, the modes of polarized growth, and the roles of "minor" tooth germ cell populations such as the inner dental epithelium-associated stratum intermedium. In our Preliminary Studies below, we now show that (a) Sonic hedgehog (SHH) is expressed in stratum intermedium and associated preameloblasts at late-bell and crown stages, and (b) treatment of tooth germs in organ culture with antibodies against SHH or SHH antisense oligonucleotides inhibits enamel organ growth, mesenchymal condensation, and cytodifferentiation of ameloblasts and odontoblasts. We show for the first time that stratum intermedium undergoes striking spatio-temporal changes in structure and phenotype during odontogenesis involving transient multilayering and SHH expression; reciprocal events occur in the associated preameloblast layer. These and additional Preliminary Studies detailed below lead us to two central hypotheses: (A) SHH is a signaling molecule produced by the dental lamina first and stratum intermedium thereafter, which is needed for tooth germ initiation and early morphogenetic events; and (B) SHH mediates epithelial- epithelial interactions between stratum intermedium and inner dental epithelium at later stages, which are needed for ameloblast terminal differentiation. Specifically , we propose to (a) determine the mechanisms of SHH signaling during odontogenesis, (b) analyze how stratum intermedium structure and phenotype change during odontogenesis, and (c) determine whether and how stratum intermedium induces and regulates ameloblast cell differentiation. To achieve these goals, we will make use of organ cultures, tissue transplantations, in situ hybridization, immunological approaches, recombinant protein preparation, and retrovirally-driven ectopic gene expression. The results of the project will provide key information on morphogenetic and cellular signals responsible for initiation and progression of odontogenesis.